Aging is a complex process that involves metabolic and physiologic changes that lead to an increasing susceptibility to disease and ultimately death. In order to address the basis for this patent, an explanation of several of the major scientific hypotheses explaining aging will be discussed. There are many theories to explain the aging process. However, three leading theories have the greatest scientific support and include: the membrane hypothesis of aging (MHA), the telomerase theory of aging, and the dysdifferentiation hypothesis of aging.
The membrane hypothesis of aging (MHA), also called the mitochondrial clock theory of aging, is based upon the progressive accumulation of oxidative damage and is directly related to this patent. This progressive damage occurs secondary to the action of reactive oxygen species (ROS) also known as free radicals which are generated in increasing quantities with age.1,2 ROS are known to damage DNA in general and mitochondrial DNA in specific, as well as cells and tissue. The mitochondrial DNA damage leads to reduced capacity for energy generation within the mitochondria and ultimately causes aging and death. This is the premise for the use of powerful antioxidants to perhaps slow the processes of aging.
The mitochondrion is a tiny structure inside a cell and is the primary generator of energy, in the form of adenosine tri-phosphate (ATP). Mitochondria have their own DNA which determines all of their functions. The mitochondrial DNA (mtDNA) is made up of 16569 base pairs that, when completely intact, makes energy for the body. However, subtle changes in the MtDNA have dramatic effects on mitochondrial function and energy production. Research in our laboratory, and in several others around the world, has identified a specific deletion (or elimination) in mitochondrial DNA that is known to occur in response to aging. It is called the common aging deletion and consists of 4977 base pairs. There are other MtDNA deletions that can occur in response to aging, such as the 520 bp deletion, etc. It is not difficult to comprehend that if you remove approximately one-third of the mitochondrial DNA you will have significant problems with energy generation. It has been found that even minor amounts of this deletion severely alter energy production and cellular function.
Studies have demonstrated an age-dependent increase in the presence of the common mitochondrial deletion (MtDNA4977 in human; MtDNA4834 in rat).3 Specifically, the common aging deletion was identified in one of fifteen young rats, while eleven of fourteen aged rats had the MtDNA deletion. The aged rats also had hearing loss, and even more interesting is that the three aged rats without the deletion had better hearing when compared to the eleven with the deletion. Additionally, we were able to study mitochondrial function in aged rats and humans, it is significantly reduced compared to the young subjects. Human studies have revealed the presence of this MtDNA deletion in white blood cells of patients with age-related hearing loss more often than in control patients.4 Two other human studies have identified the common aging deletion (MtDNA4977) in patients with age-related hearing loss more than in control subjects.5,6 
It is proposed to use this sensitive molecular biologic test to study MtDNA deletions and determine, with accuracy, an individual's “molecular age”. Preliminary evidence and logic predicts that even though two people may have the same chronologic age, that due to variations in lifestyle, diet, socio-economic factors and genetics, their molecular age may well be very different. For example: There are two forty year old men: One lives in Northern Michigan (at sea level) has an excellent diet, exercises regularly, supplements with specific nutrients, doesn't smoke or spend much time in the sun. Additionally, this Michigan native has a body mass index of 22 (normal=<25). Contrast this to another 40 year old man who lives in Colorado (about 5000 feet above sea level, this provides for more ionizing radiation), has a poor diet, rarely exercises, doesn't use nutritional supplements, smokes a half pack of cigarettes per day and is always out in the sun. Additionally, his BMI is 32 (considered obese). Even though both are 40 years old, analysis of their mitochondria shows vast differences with 10-200 fold increases in the MtDNA deletion in the gentleman from Colorado. In essence, the man from Colorado has more rapid aging and in reality has the mitochondria of a 65 year old. This information is a wakeup call to alter one's lifestyle immediately. This test provides critical information regarding one's molecular age and an indirect measure of long-term ROS damage.
It is known that certain tissues are more susceptible to oxidative damage (damage from free radicals) and reduced energy supply. This is particularly true for tissues that no longer make new cells. For example, brain, eye, inner ear, and all muscle can accumulate high amounts of these deletions and they become more susceptible to free radical damage than other tissues. Thus, increased oxidative damage that is associated with aging preferentially affects these tissues.
There are two other leading theories of aging: (1) the telomerase theory of aging and (2) the dysdifferentiation theory of aging. The end of a chromosome is made up of a structure called the telosome. The tip of the telosome is a region of repeating DNA sequences and proteins called the telomere. The telomerase theory of aging suggests that there is a reduction in telomere length over time.7 Another way to look at this is to consider the telosome as similar to the tail of a rattlesnake. There are a finite number of rings on a telosome (or a rattlesnake) and the theory suggests that each time the telosome reproduces one ring is lost. When there are only a few rings of the telosome left, death is imminent. Interestingly, the activation of the enzyme responsible for making these rings disappear (telomerase enzyme) can be manipulated experimentally. However, it has already been found that cancer alters the telomerase enzyme, thereby becoming immortal. It is felt that special genes, called viral oncogenes, may produce immortality of a cell or tissue by activating telomerase, thus effectively preventing telomere shortening and sustaining cellular growth of tumors.8 Although many aspects of telomerase activity remain undefined, it has been hypothesized that the balance between telomere shortening and telomerase activity may underlie cellular aging processes. Furthermore, caution must be exercised when these genes are manipulated, because of the potential to trigger cancerous change.
The dysdifferentiation hypothesis suggests that there is a preprogrammed activation of genes that are deleterious to the cell and lead to activation of enzymes and reactions that are responsible for age-related changes. This line of reasoning was, in part, brought to the forefront from work elaborating control mechanisms of aging in the earthworm. Two main genes, Bax and BCl2, have essential roles in cellular aging and immortality respectively. Scientists were able to increase the lifespan of the common earthworm by 30-40% by increasing the activity of the BCl2 gene. However, once again, it has been shown that several cancers become immortal precisely by up-regulating the BCl2 gene.
The process of aging is associated with many molecular, biochemical and physiological changes including increases in DNA damage, reduction in mitochondrial function, decreases in cellular water concentrations, ionic changes, and decreased elasticity of cellular membranes. One contributing factor to this process is altered vascular characteristics, such as reduced flow and vascular plasticity as well as increased vascular permeability.9,10 Atherosclerosis and high lipids and cholesterol further affect these situations and reduce the overall blood flow to many tissues in the body. These age-related changes result in reductions in oxygen and nutrient delivery and in waste elimination.11-14 These physiologic inefficiencies favor the production of ROS. Furthermore, there is support in the literature for age-associated reduction in enzymes that protect from ROM damage including superoxide dismutase, catalase and glutathione.15-17 Collectively, these changes enhance the generation of ROS.
One of the most important factors in aging is the level of oxidative stress. Oxidative stress occurs when the usual balance between reactive oxygen species and antioxidants is disturbed. Each individual's level of oxidative stress is different and depends on a number of factors including fitness, genetics, disease and metabolic rate. There is a continuing need for a method to formulate a molecular age of an individual based on the individual's physiological state and level of oxidative stress in order to provide a basis and motivation for medical treatment and lifestyle change.